USPTO Patents Application 09993647

Europaisches Patentamt
European Patent Office
Office europeen des brevets

© Publication number:

0 502 504 A1

EUROPEAN PATENT APPLICATION

© Application number: 92103691.9
0 Date of filing: 04.03.92

© int. CI. 5 : C12Q 1/00, G01N 27/404

®

Priority: 04.03.91 JP 37259/91

© Inventor: Yoshioka, Toshihiko

04.03.91 JP 37261/91

4-15-11-302, Shinmori, Asahi-ku

02.09.91 JP 221402/91

Osaka-shi, Osaka(JP)

©

Date of publication of application:

Inventor: Nankai, Shiro

09.09.92 Bulletin 92/37

4-50-12, Nasuzukuri
Hirakata-shi, Osaka(JP)

©

Designated Contracting States:
DE FR GB IT

©

Applicant: MATSUSHITA ELECTRIC
INDUSTRIAL CO., LTD

© Representative: Marx, Lothar, Dr. et al
Patentanwalte Schwabe, Sandmair, Marx

1006, Oaza Kadoma, Kadoma-shi

Stuntzstrasse 16

Osaka 571 (JP)

W-8000 Munchen 80(DE)

© A biosensor utilizing enzyme and a method for producing the same.

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IT)

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© The biosensor of the present application deter-
mines an analyte (eq fructose) contained in a sample
liquid by reducing electron acceptors using electrons
generated in a reaction of the analyte with an en-
zyme and then by measuring the reduced amount of
the electron acceptors electrochemically. The
biosensor has an electrical insulating substrate (1),
an electrode system (14) including at least a working
electrode (4) and a counter electrode (5), a reaction
layer (15) including the enzyme (7) provided on the
electrode system and a hydrogen ion concentration
(pH) control layer (11), and the reaction layer is in
contact with the electrode system. According to the
present application, the pH of the sample can be
optimised for the type of the enzyme contained in
the reaction layer, without prior addition of pH buffer
to the sample liquid. Thus, the specific analyte con-
tained in the sample liquid can be easily determined
with accuracy and speed.

Fig,1

Rank Xerox (UK) Business Services

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EP 0 502 504 A1

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BACKGROUND OF THE INVENTION

1. Field of the Invention:

The present invention relates to a biosensor
that can easily quantify a specific component in a
sample liquid with accuracy and speed, and a
method for producing the same, and more particu-
larly to a biosensor for quantifying a specific com-
ponent in a sample liquid by reducing electron
acceptors using electrons generated in the reaction
of the specific component in the sample liquid to
enzyme that specifically reacts to the component,
and then by electrochemically measuring the re-
duced amount of electron acceptors, and a method
for producing the same.

2. Description of the Prior Art:

Various types of biosensors utilizing specific
catalyses of enzyme have been recently devel-
oped. A saccharide biosensor will be described as
an example of such biosensors as follows:

The optical rotation method, the colorimetric
method, the reductimetry method and other meth-
ods using different kinds of chromatographies have
been developed as methods for quantitative analy-
sis of saccharides. However, none of these meth-
ods can provide high accuracy due to the relatively
low specificity against saccharides. Additionally,
the optical rotation method is easy to operate but is
largely influenced by the operating temperature.
Therefore, it is not appropriate for common use at
home and the like.

The saccharides contained in fruit are generally
assessed as saccharine degrees. A refractometer
of the light refraction system is often used for
quantifying the saccharine degree. This refracto-
meter functions by utilizing change of the light
refractive index caused by liquid concentration.
Therefore, the refractometer of the light refraction
system is influenced by all the components dis-
solved in the sample liquid, for example, by or-
ganic acid such as citric acid or malic acid con-
tained in fruit juice in a large amount when a
saccharide in fruit is quantified. Thus, accurate
quantification by this refractometer is impossible.

A glucose sensor will now be described as an
example of a biosensor used in a clinical field.

A conventional method for quantifying glucose
contained in blood is to centrifuge blood taken from
a patient and then to measure the thus obtained
blood plasma. This method requires a lot of time
as well as labor. Therefore, a sensor that can
directly measure glucose in blood obtained from a
patient has been desired.

A sensor similar to a test paper for urinalysis
has been developed as a simple glucose sensor.

This glucose sensor comprises a support in a stick
shape and a holder fixed to the support. The holder
includes an enzyme reacting only to glucose and a
dye the color of which is changed by reacting with

5 a production of the enzyme reaction. Blood is
dropped on the support of the glucose sensor and
the change of the dye after a predetermined period
of time of the dropping is visually observed or
optically measured, whereby the content of glucose

io in the blood can be measured. However, the quan-
tifying method using this glucose sensor has low
accuracy due to the interference by the colored
materials in the blood.

Japanese Laid-Open Patent Publication No. 1-

75 114747 discloses the following glucose sensor with
high accuracy as a method for quantifying a spe-
cific component in a sample liquid from a living
body such as blood without diluting or stirring the
sample liquid:

20 As shown in Figure 7 this glucose sensor com-

prises an electrical insulating substrate 51, an elec-
trode system 54 including a working electrode 52
and a counter electrode 53 formed on the insulat-
ing substrate 51 by screen printing, an electrical

25 insulating layer 55 formed on the insulating sub-
strate 51, an adhesive structure 56 provided on the
electrical insulating layer 55, a filtration layer 57
supported by the adhesive structure 56, a holding
frame 58 provided on the filtration layer 57, and an

30 electron acceptor holding layer 59, an enzyme
holding layer 60, a buffering salt holding layer 61
and an expansion layer 62 which are supported by
the holding frame 58. A space 63 for containing a
sample liquid is formed between the electrode sys-

35 tern 54 and the filtration layer 57.

The filtration layer 57 is made from a porous
polycarbonate film. The electron acceptor holding
layer 59, the enzyme holding layer 60 and the
buffering salt holding layer 61 use porous cellulose

40 as supports.

The operation of such a glucose sensor is as
follows: The sample liquid dropped on the expan-
sion layer 62 is adjusted to pH that provides the
most stable enzyme activity by the action of the

45 buffering salt in the buffering salt holding layer 61.
Then, in the enzyme holding layer 60, the glucose
oxidase in the enzyme holding layer 60 and the
glucose in the sample liquid specifically react to
each other. The potassium ferricyanide in the elec-

50 tron acceptor holding layer 59 is then reduced to
become potassium ferrocyanide by electrons gen-
erated in the above reaction. The amount of the
potassium ferrocyanide generated at this time is in
proportion to the glucose concentration in the sam-

55 pie liquid. Next, materials with larger molecular
weight such as protein are filtered in the filtration
layer 57, and the filtered liquid drops in the space
63 above the electrode system 54. Thus the

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EP 0 502 504 A1

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amount of potassium ferrocyanide in the liquid can
be measured by measuring the oxidation current of
the liquid by the electrode system 54, thereby
measuring the glucose concentration.

In the conventional glucose sensor with the
above-mentioned structure, frothing may remain in
the space due to the inconstant flow of the liquid in
the space 63, which influences the measured value
of the glucose concentration.

Furthermore, since the buffering salt holding
layer 61 is in contact with the enzyme holding layer
60, the buffering salt and the enzyme are mixed on
the interface between the two layers when the
glucose sensor absorbed moisture, thereby deterio-
rating the enzyme activity by the chemical inter-
action. As a result, the glucose sensor of this type
is hard to store in a stable condition.

Moreover, since insoluble porous materials are
used as supports of the filtration layer 57 and each
of the holding layers 59, 60, and 61 in the conven-
tional glucose sensor, the sample liquid supplied to
the glucose sensor is required to pass through
each of the porous materials before reaching the
electrode system 54. Therefore, the sensor has
disadvantages that it may take longer time to ob-
tain the reaction and/or that the response values
may be inconstant due to the inconstant reaction
time. Additionally, the glucose sensor has so many
steps in its production, including such a compli-
cated step as to assembly a plurality of porous
materials, that it is difficult to produce it at a low
cost.

SUMMARY OF THE INVENTION

The biosensor of this invention for quantifying a
substrate contained in a sample liquid by reducing
electron acceptors using electrons generated in a
reaction of the substrate to enzyme and then by
measuring the reduced amount of the electron ac-
ceptors electrochemically, which overcomes the
above-discussed and numerous other disadvan-
tages and deficiencies of the prior art, comprises
an electrical insulating substrate, an electrode sys-
tem including at least a working electrode and a
counter electrode which are formed on the insulat-
ing substrate, a reaction layer including the en-
zyme provided on the electrode system, and a
hydrogen ion concentration control layer, wherein
the reaction layer is in contact with the electrode
system.

In a preferred embodiment, the reaction layer
further includes hydrophilic polymer and electron
acceptors.

In a preferred embodiment, the reaction layer
further includes hydrophilic polymer and the hy-
drogen ion concentration control layer includes
electron acceptors.

In a preferred embodiment, the reaction layer
is formed by laminating a first layer including hy-
drophilic polymer and enzyme, a second layer in-
cluding hydrophilic polymer and a third layer in-

5 eluding electron acceptors in this order.

In a preferred embodiment, the hydrogen ion
concentration control layer is provided on the elec-
trode system.

In a preferred embodiment, the reaction layer

70 further includes hydrophilic polymer and electron
acceptors, and a hydrophilic polymer layer is pro-
vided between the reaction layer and the hydrogen
ion concentration control layer.

In a preferred embodiment, the reaction layer

75 comprises a first layer including hydrophilic poly-
mer and electron acceptors and a second layer
including enzyme laminated on the first layer, and
the hydrogen ion concentration control layer is
provided on the second layer.

20 In a preferred embodiment, the electrode sys-

tem is mainly formed from carbon.

In a preferred embodiment, the hydrogen ion
concentration control layer includes buffering salt
selected from a group consisting of potassium

25 biphosphate-dipotassium phosphate, potassium
biphosphate-disodium phosphate, citric acid-dis-
odium phosphate, citric acid-trisodium citrate, po-
tassium bicitrate-sodium hydroxide and maleic acid
monosodium salt-sodium hydroxide.

30 In a preferred embodiment, the hydrophilic

polymer is selected from a group consisting of
polyvinyl alcohol and cellulose derivatives, con-
cretely, hydroxy proryl cellulose, methyl cellulose,
ethyl cellulose, ethyl hydroxyethyl cellulose and

35 carboxy methyl ethyl cellulose, polyvinylpyr-
rolidone, gelatin or its derivatives, (meth)acrylic
acid or its salts, starch or its derivatives and maleic
anhydride or its salts.

In a preferred embodiment, the enzyme is se-

40 lected from a group consisting of fructose de-
hydrogenase, invertase, mutarotase, glucose ox-
idase, alcohol oxidase, lactase oxidase, lactase de-
hydrogenase, cholesterol oxidase, xanthine oxidase
and amino acid oxidase.

45 In a preferred embodiment, the electron accep-

tor is selected from a group consisting of potas-
sium ferricyanide, p-benzoquinone,
phenazinemethosulfate and ferrocene.

Alternately, the present invention provides a

so method for producing a biosensor for quantifying a
substrate contained in a sample liquid by reducing
electron acceptors using electrons generated in a
reaction of the substrate to enzyme and then by
measuring the reduced amount of the electron ac-

55 ceptors electrochemically, the method comprising
the steps of:

firstly, providing an electrode system including
at least a working electrode and a counter elec-

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trode on an electrical insulating substrate;

secondly, forming a hydrogen ion concentra-
tion control layer including electron acceptors on
the insulating substrate; and

finally, forming a reaction layer including hy-
drophilic polymer and enzyme on the electrode
system.

Thus, the invention described herein makes
possible the objectives of providing (1) a biosensor
in which the hydrogen ion concentration of a sam-
ple liquid can be made most adequate in accor-
dance with the type of enzyme contained in a
reaction layer without prior adjustment of the hy-
drogen ion concentration in the sample liquid, (2) a
biosensor which can easily quantify a specific sub-
strate contained in a sample liquid with accuracy
and speed, (3) a biosensor in which the reliability in
performance thereof while being stored can be
improved by separating buffering salt contained in
a hydrogen ion concentration control layer from
enzyme contained in a reaction layer by a hydro-
philic polymer layer, (4) a method for producing a
biosensor in a short period of time by heating
layers such as a hydrogen ion concentration con-
trol layer including electron acceptors, and (5) a
method for producing a biosensor, in which crystal
diameters of electron acceptors contained in a lay-
er can be optionally controlled by controlling the
temperature in heating the layer.

BRIEF DESCRIPTION OF THE DRAWINGS

This invention may be better understood and
its numerous objects and advantages will become
apparent to those skilled in the art by reference to
the accompanying drawings as follows:

Figure 1 is a sectional view of a base of a

fructose sensor according to an example of a

biosensor of the present invention;

Figures 2 and 3 are exploded perspective views

of the fructose sensor of Figure 1;

Figure 4 is a diagram showing the response

characteristics of the fructose sensor;

Figure 5 is a sectional view of a base of a

fructose sensor according to another example of

the present invention;

Figure 6 is an exploded perspective view of the
fructose sensor of Figure 5; and
Figure 7 is a diagram showing an example of a
conventional biosensor.

DESCRIPTION OF THE PREFERRED EMBODI-
MENTS

A biosensor according to the present invention
comprises an electrical insulating substrate, an
electrode system including a working electrode and
a counter electrode provided on the insulating sub-

strate, a reaction layer including enzyme provided
on the electrode system and a hydrogen ion con-
centration control layer provided on the insulating
substrate.

5 According to the present invention, the enzyme

is not influenced by the buffering salt included in
the hydrogen ion concentration control layer be-
cause the reaction layer including the enzyme is
formed separately from the hydrogen ion concen-

io tration control layer. Therefore, the contained en-
zyme can be kept in a stable condition when the
biosensor is stored.

Generally, the pH of a sample liquid is not
necessarily the same as that of the specified en-

75 zyme in the reaction layer that provides the highest
enzyme activity. In accordance with the present
invention, the pH of the sample liquid can approxi-
mate to the value which provides the highest activ-
ity of the enzyme by allowing the sample liquid

20 supplied to the sensor to reach the hydrogen ion
concentration control layer. As a result, the sensor
can be simply operated because there is no need
to adjust the pH of the sample liquid by a buffer
solution or the like.

25 A method for producing a biosensor of the

present invention comprises steps of providing an
electrode system including at least a working elec-
trode and a counter electrode on an electrical in-
sulating substrate, forming a hydrogen ion con-

30 centration control layer including at least electron
acceptors, and finally forming a reaction layer in-
cluding at least hydrophilic polymer and enzyme
on the electrode system.

Generally the activity of enzyme is largely re-

35 duced in a treatment at a temperature of several
tens degrees centigrade. According to the method
of the present invention, the reaction layer is not
treated at a high temperature that may inhibit the
enzyme activity, since the hydrogen ion concentra-

40 tion control layer including the electron acceptors
is formed prior to the formation of the reaction
layer including the enzyme. Therefore, it is possi-
ble to set the heat treatment condition of the hy-
drogen ion concentration control layer optionally

45 depending upon the objectives of the biosensors.
For example, the particle diameter of the crystal of
the electron acceptor or the drying condition there-
of can be controlled so as to be most appropriate,
by making shorter the heating time of the hydrogen

50 ion concentration control layer and/or by controlling
the heating temperature of the hydrogen ion con-
centration control layer.

The use of the biosensor of the present inven-
tion depends upon the substrate (the specified

55 component), that is, the object to be measured,
contained in the sample liquid. The sensor may be
used as, for example, a fructose sensor, a sucrose
sensor, a glucose sensor, an alcohol sensor, a

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lactic acid sensor, a cholesterol sensor and an
amino acid sensor.

The enzyme used in the present invention de-
pends upon the substrate contained in the sample
liquid and is not limited. The usable enzyme are,
for example, fructose dehydrogenase, invertase,
mutarotase, glucose oxidase, alcohol oxidase, lac-
tase oxidase, lactase dehydrogenase, cholesterol
oxidase, xanthine oxidase and amino acid oxidase.

Any buffer solution with pH that provides the
highest activity of enzyme used for producing a
biosensor may be chosen in order to form the
hydrogen ion concentration control layer. The us-
able buffer solutions are, for example, a phosphate
buffer solution, a Mcllvaine buffer solution, citric
acid-trisodium citrate, potassium bicitrate-sodium
hydroxide and maleic acid monosodium salt-so-
dium hydroxide. Further, a buffer solution which is
liquid at an ordinary temperature such as an acetic
acid-sodium acetate buffer solution may be used.
In this case, the hydrogen ion concentration control
layer may be formed together with a polymer with
a long shelf life.

The hydrophilic polymer used in this invention
is not limited. The usable hydrophilic polymers are,
for example, polyvinyl alcohol and cellulose deriva-
tives, concretely, hydroxy proryl cellulose, methyl
cellulose, ethyl cellulose, ethyl hydroxyethyl cel-
lulose and carboxy methyl ethyl cellulose, poly-
vinylpyrrolidone, gelatin or its derivatives, (meth)-
acrylic acid or its salts, starch or its derivatives and
maleic anhydride or its salts.

As for the electron acceptors in this invention,
potassium ferricyanide, p-benzoquinone,
phenazinemethosulfate and ferrocene may be
used.

The above-described electrode system is not
limited to a two-electrode system having only a
working electrode and a counter electrode. For
example, a three-electrode system, including an
additional reference electrode, may be used, so
that more precise values are obtainable.

EXAMPLES

Throughout the drawings mentioned in the fol-
lowing description of the examples, the same ele-
ment has a common reference numeral. Part of the
description of the producing procedure is omitted
as occasion demands.

Example 1

A fructose sensor will now be described as an
example of a biosensor.

A fructose sensor of the present invention com-
prises a base 10, a spacer 20 adhered to the base
10 and a cover 30 adhered to the spacer 20 as

shown in Figures 1 to 3. Figure 1 shows a sectional
view of the base 10 of the fructose sensor, and
Figure 2 shows an exploded perspective view of
the fructose sensor with a reaction layer 15 and a

s hydrogen ion concentration control layer 11 re-
moved from the base 10. Figure 3 is also an
exploded perspective view of the fructose sensor.

Silver paste was printed by means of screen
printing on an electrical insulating substrate 1 made

70 from polyethylene terephthalate to form leads 2
and 3. Then, an electrode system 14 (a working
electrode 4 and a counter electrode 5) made from
conductive carbon paste including resin binder and
an insulating layer 6 made from insulating paste

75 were formed by screen printing. The insulating
layer 6 was covering the leads 2 and 3 except for
connecting portions 12 and 13 with a connector so
that the predetermined areas of the working elec-
trode 4 and the counter electrode 5 were exposed

20 respectively.

Then, the exposed portions of the working
electrode 4 and the counter electrode 5 were
ground and heat treated for 4 hours at 100 6 C in
the air.

25 After forming the electrode system 14 compris-

ing the working electrode 4 and the counter elec-
trode 5 in this way, an aqueous solution including
0.5 wt% of carboxymethyl cellulose (hereinafter
called the CMC), as hydrophilic polymer, was

30 dropped on the electrode system 14 and dried, to
form a CMC layer. Then an aqueous solution of
fructose dehydrogenase (manufactured by Toyo
Boseki Kabushiki Kaisha; EC1 .1 .99.1 1), as enzyme,
was dropped so as to cover the CMC layer and

35 dried, to form a CMC-fructose dehydrogenase layer
7. In this CMC-fructose dehydrogenase layer 7, the
CMC layer and the fructose dehydrogenase layer
were mixed only by a thickness of several microns
on the interface therebetween, namely, both layers

40 were not completely mixed.

An ethanol solution including 0.5 wt% of poly-
vinylpyrrolidone (hereinafter called the PVP), as
hydrophilic polymer, was dropped so as to com-
pletely cover the CMC-fructose dehydrogenase lay-

45 er 7 and dried, to form a PVP layer 8.

When fruit juice including solid elements such
as sarcocarp is used as a sample liquid, the solid
elements are stopped by the PVP layer 8 provided
on the CMC-fructose dehydrogenase layer 7, re-
st? suiting in the prevention of the solid elements from
being adsorbed to the surface of the electrode
system 14. As a result, the response function of the
sensor is prevented from being deteriorated. Fur-
thermore, since the PVP layer 8 separates the

55 fructose dehydrogenase contained in the CMC-
fructose dehydrogenase layer 7 from potassium
ferricyanide described below, the conservative and
stable properties of the sensor can be remarkably

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improved.

Dispersing liquid, with crystals of potassium
ferricyanide (the particle diameter: 0.5 - 4 urn), that
is, electron acceptors, dispersed in a toluene solu-
tion including 0.5 wt% of lecithin as a dispersing
agent was dropped and dried at room temperature,
to form a potassium ferricyanide-lecithin layer 9 on
the PVP layer 8.

Thus the reaction layer 15 comprising the
CMC-fructose dehydrogenase layer 7, the PVP lay-
er 8 and the potassium ferricyanide-lecithin layer 9
was formed.

Then, 3 ul of a phosphate buffer solution (a
mixture of 0.2 M of K 2 HPO* and 0.2 M of KH2PO4,
pH = 4.5) was dropped on a portion between the
reaction layer 15 and the tip portion of the insulat-
ing substrate corresponding to a sample supply
port 21, and dried, to form a hydrogen ion con-
centration control layer 11 as shown in Figures 1
and 3.

The enzyme used in this fructose sensor, that
is, fructose dehydrogenase, shows the highest ac-
tivity when pH is 4.5 (at 37 6 C). Since a fructose
standard solution is almost neutral, when the stan-
dard solution reaches the hydrogen ion concentra-
tion control layer 11, the pH of the solution be-
comes 4.5, thereby making the enzyme activity the
highest. Alternately, when the sample liquid is acid
or alkaline, the pH of the liquid is adjusted to be
approximately 4.5, when the liquid reaches the
hydrogen ion concentration control layer 11. There-
fore, in the liquid that has passed the hydrogen ion
concentration control layer 11, the enzyme activity
will become higher as compared with in the
originally supplied sample liquid.

Furthermore, the enzyme in the reaction layer
15 is separated from the hydrogen ion concentra-
tion control layer 11 by forming the hydrogen ion
concentration control layer 11 away from the reac-
tion layer 15, thereby the preservative property of
the sensor is improved.

After forming the base 10 including the reac-
tion layer 15 and the hydrogen ion concentration
control layer 11 in the above-described way, the
base 10, the spacer 20 and the cover 30 were
laminated to be adhered as shown in Figures 2 and
3 with dashed lines. A reservoir for a sample liquid
was formed as a space defined by the base 10, the
wall of a groove 22 of the spacer 20 and the cover
30. As a result the sample liquid supply port 21
was formed at one end of the reservoir. An air port
31 was formed on a portion of the cover 30 oppos-
ing to the other end of the reservoir.

A sample liquid is supplied into the reservoir
and introduced toward the reaction layer 15 only
by allowing the sample liquid to contact the sample
supply port 21 formed at the tip of the sensor.
Since the supply amount of the sample liquid de-

pends upon the volume of the reservoir, prior quan-
tification of the liquid is not necessary. Further-
more, evaporation of the sample liquid during the
measurement can be minimized, resulting in a

5 measurement with high accuracy.

The thus produced fructose sensor was sup-
plied with 3 ul of a fructose standard solution as a
sample liquid from the sample supply port 21. Two
minutes after the supply, a pulse voltage of + 0.5 V

10 on the basis of the voltage at the counter electrode
5 was applied to the working electrode 4. Then the
anodic current value of five seconds after the ap-
plication was measured.

When the sample liquid having obtained the

75 desired pH after passing the hydrogen ion con-
centration control layer 11 reaches the reaction
layer 15, the potassium ferricyanide-lecithin layer
9, the PVP layer 8 and the CMC-fructose de-
hydrogenase layer 7 are dissolved into the sample

20 liquid successively in this order. The fructose con-
tained in the sample liquid is oxidized by fructose
dehydrogenase, and at this point the potassium
ferricyanide is reduced to potassium ferrocyanide
by shifted electrons. Then, an oxidation current

25 corresponding to the concentration of the gener-
ated potassium ferrocyanide is caused by the ap-
plication of the above pulse voltage. The current
value corresponds to the concentration of the sub-
strate, that is, fructose in this case.

30 Figure 4 shows the response property mea-

sured two minutes after the supply of the sample
liquid by using the fructose sensor of this example.
In the graph, the curve a shows responses of the
fructose sensor of this example. The curve b

35 shows responses of a comparative fructose sensor,
in which the hydrogen ion concentration control
layer 11 is removed from the base 10 of the
fructose sensor, obtained in the same way as in
this example.

40 As is evident from Figure 4, in the fructose

sensor of this example, the relation between the
fructose concentration and the sensor response
corresponded proportionally up to 25 mM of fruc-
tose, the substrate. On the other hand, in the

45 fructose sensor without a hydrogen ion concentra-
tion control layer, the fructose concentration cor-
responded to the sensor response only up to 10
mM of fructose.

In Figure 4, in the range where the correspond-

50 ing relation between the fructose concentration and
the sensor response is shown as a straight line, the
response current value obtained by the fructose
sensor of this example was larger by about 20%
than that of the comparative fructose sensor with-

55 out the hydrogen ion concentration control layer
11

This example shows that a biosensor with a
wide measuring range and a high sensitivity can be

6

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EP 0 502 504 A1

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obtained by providing the hydrogen ion concentra-
tion control layer 11 on the base 10.

Furthermore, in the fructose sensor of this ex-
ample, when the measuring time was set to be one
minute, the relation that can be shown as a straight
line was obtained up to 15mM of fructose. The
response repeatability of the sensor was also ex-
cellent. For example, the coefficient of variation
(the CV value) in using thirty sensors as to the
same sample liquid was 7% or less.

Example 2

An electrode system 14 comprising a working
electrode 4 and a counter electrode 5 as shown in
Figure 1 was formed on an insulating substrate 1
made from polyethylene terephthalate by means of
screen printing in the same manner as in Example
1, and was ground and heat treated. A mixed
aqueous solution including 0.5 wt% of CMC, fruc-
tose dehydrogenase and potassium ferricyanide
was dropped on the electrode system 14 and was
dried for 10 minutes in a warm-air drier at 40° C, to
form a reaction layer 15.

The production procedure of a biosensor can
be simplified by forming the reaction layer 15 by
dropping and drying a mixed solution of hydrophilic
polymer, enzyme and electron acceptors on the
electrode system 14 in the above way.

Then, 3 llI of a Mcllvaine buffer solution (a
mixture of 0.1 M of citric acid and 0.2 M of dis-
odium phosphate, pH = 4.5) was dropped on a
portion between the reaction layer 15 and a tip
portion of the insulating substrate 1 corresponding
to a sample supply port 21 of the sensor, and was
dried to form a hydrogen ion concentration control
layer 11. The thus obtained base 10, a spacer 20
and a cover 30 were laminated to be adhered as in
Example 1 as shown in Figures 2 and 3 with
dashed lines.

Three ml of a fructose standard solution was
supplied to the thus obtained fructose sensor from
the sample supply port 21. Two minutes after the
supply, a pulse voltage of +0.5 V on the basis of
the voltage of the counter electrode 5 was applied
to the working electrode 4. The anodic current
value 5 seconds after the application was mea-
sured, resulting in obtaining a response current
value corresponding to the fructose concentration
in the sample liquid. Alternately, the sample liquid
was supplied in the same manner and the voltage
was applied after 90 seconds. The obtained re-
sponse current was almost the same as that ob-
tained after 2 minutes. Moreover, the coefficient of
variation measured in using 30 sensors as to the
same sample liquid was 5% or less. Thus, the
repeatability was also excellent.

In the fructose sensor of this example, the

fructose dehydrogenase mixed with potassium fer-
ricyanide exists in the reaction layer 15. Therefore,
both the fructose dehydrogenase and the potas-
sium ferricyanide are easily dissolved into the sam-

s pie liquid rapidly and uniformly. This rapid reaction
in the mixture reduces the time required for mea-
suring and makes the response current constant.

The response current obtained by the fructose
sensor of this example was larger by about 20%

70 than that of the comparative fructose sensor with-
out a hydrogen ion concentration control layer as
was used in Example 1 .

Example 3

75

An electrode system 14 comprising a working
electrode 4 and a counter electrode 5 as shown in
Figure 1 was formed on an insulating substrate 1
made from polyethylene terephthalate by means of

20 screen printing in the same manner as in Example
1, and was ground and heat treated. Then, 3 i±\ of
a phosphate buffer solution (a mixture of 0.2 M of
K2HPO4 and 0.2 M of KH2PO4, pH = 4.5) includ-
ing potassium ferricyanide was dropped on a por-

25 tion between the electrode system 14 and the tip
portion of the insulating substrate corresponding to
a sample supply port 21, and was dried at 100°C
for 5 minutes by heating as shown in Figures 1 and
3, to form a hydrogen ion concentration control

30 layer 11 including potassium ferricyanide.

The time required for drying the hydrogen ion
concentration control layer 11 depends upon the
temperature at which the layer is heated. There-
fore, the crystal diameter of the potassium ferricya-

35 nide contained in the hydrogen ion concentration
control layer 11 can be controlled by the heat
treatment condition. The crystal diameter of the
potassium ferricyanide becomes smaller when the
drying time is shorter, resulting in increasing the

40 solubility speed into the sample liquid. Thus the
response speed of the sensor can be increased.
On the other hand, when the potassium ferricya-
nide coexists with the enzyme in the reaction layer
15, the enzyme activity is reduced by heating the

45 reaction layer 15 at a high temperature. Therefore,
the heating temperature of the layer 15 can not be
set freely.

Furthermore, as in the present example, when
the enzyme in the reaction layer 15 is separated

50 from the potassium ferricyanide by containing the
potassium ferricyanide in the hydrogen ion con-
centration control layer 11, the shelf life of the
sensor can be remarkably increased.

Then, a mixed aqueous solution including 0.5

55 wt% of CMC and fructose dehydrogenase was
dropped on the electrode system 14, and was
dried in a warm-air drier at 40° C for 10 minutes, to
form the reaction layer 15. The thus formed base

7

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10, a spacer 20 and a cover 30 were laminated to
form a fructose sensor.

The fructose sensor produced in the above
way was supplied with 3 u\ of a fructose standard
solution from the sample supply port 21. The sen-
sor response was measured 2 minutes after the
supply in the some manner as in Example 1. As a
result, the obtained current value corresponded to
the fructose concentration in the sample liquid.
Alternately, when the voltage was applied 90 sec-
onds after supplying the sample liquid as de-
scribed above, the response current value was
approximately the same as that obtained after 2
minutes. This is because the potassium ferricya-
nide in the sample liquid was rapidly dissolved due
to the smaller crystal diameters of the potassium
ferricyanide (0.05 - 0.3 jum).

The response current obtained by the fructose
sensor of this example was larger by about 20%
than that of the comparative fructose sensor with-
out a hydrogen ion concentration control layer 11
as was in Example 1 .

Example 4

A sucrose sensor will now be described as
another example of a biosensor.

An electrode system 14 comprising a working
electrode 4 and a counter electrode 5 as shown in
Figure 1 was formed on an insulating substrate 1
made from polyethylene terephthalate, and was
ground and heat treated. Then, an aqueous solution
including 0.5 wt% of CMC was dropped on the
electrode system 14 and was dried to form a CMC
layer. Then a mixed aqueous solution including 0.5
wt% of CMC, invertase (EC 3.2.1.26), mutarotase
(EC5.1.3.3), glucose oxidase (EC1.1.3.4) and potas-
sium ferricyanide was dropped on the CMC layer
and was dried in a warm-air drier at 40 °C for 10
minutes to form a reaction layer 15.

Then a phosphate buffer solution (pH = 7.4)
was dropped on a portion between the reaction
layer 15 and the tip portion of the insulating sub-
strate 1 corresponding to a sample supply port 21,
then was dried to form a hydrogen ion concentra-
tion control layer 11. The thus obtained base 10, a
spacer 20 and a cover 30 were laminated to form a
sucrose sensor.

Three i±\ of a sucrose standard solution was
supplied to the sucrose sensor from the sample
supply port 21. Two minutes after the supply, a
pulse voltage of +0.5 V on the basis of the voltage
of the counter electrode 5 was applied to the
working electrode 4. Then the anodic current value
after 5 seconds was measured. As a result, the
obtained response current value corresponded to
the sucrose concentration in the sample liquid.

Example 5

An electrode system 14 comprising a working
electrode 4 and a counter electrode 5 was formed

5 on an insulating substrate 1 made from polyethyl-
ene terephthalate by means of screen printing as
shown in Figure 5, and was ground and heat treat-
ed. A mixed aqueous solution including 0.5 wt% of
CMC, fructose dehydrogenase and potassium fer-

io ricyanide was dropped on the electrode system 14,
and was dried in a warm-air drier at 40 °C for 10
minutes to form a CMC-fructose dehydrogenase-
potassium ferricyanide layer 16.

Then, an ethanol solution including 0.5 wt% of

75 PVP as hydrophilic polymer was dropped so as to
cover the entire CMC-fructose dehydrogenase-po-
tassium ferricyanide layer 16, and was dried to
form a PVP layer 8. 3 nl of a Mcllvaine buffer
solution (a mixture of 0.1 M of citric acid and 0.2 M

20 of disodium phosphate, pH = 4.5) was dropped on
the PVP layer 8, and was dried to form a hydrogen
ion concentration control layer 11.

In this example, the hydrogen ion concentration
control layer 11 and the PVP layer 8 are mixed

25 only by a thickness of several microns on the
interface therebetween since PVP is hydrophilic
polymer. Therefore, the CMC-fructose
dehydrogenase-potassium ferricyanide layer 16 is
separated from the hydrogen ion concentration

30 control layer 11 by the PVP layer 8. In this exam-
ple, a reaction layer 15 comprises the CMC-fruc-
tose dehydrogenase-potassium ferricyanide layer
16.

Thus, the time required for solution of the

35 reaction layer 15 into the sample liquid after the
hydrogen ion concentration control layer 11 is dis-
solved, because the hydrogen ion concentration
control layer 11 is formed on the reaction layer 15,
thereby reducing the time required for the sensor

40 response.

Furthermore, in the fructose dehydrogenase
used in this example as enzyme, the pH for allow-
ing the enzyme activity to be the highest is dif-
ferent from the pH for providing a high conser-

45 vative stability of the sensor. Therefore, in the
conventional biosensor as shown in Figure 7, the
shelf life of the sensor is deteriorated because the
sensor is under a pH condition which is different
from that providing the fructose dehydrogenase

50 with a high stability when the biosensor absorbs
moisture.

The biosensor of this example can overcome
the above-mentioned disadvantage because the
CMC-fructose dehydrogenase-potassium ferricya-
55 nide layer 16 can be separated from the hydrogen
ion concentration control layer 11 by the PVP layer
8.

Moreover, in the biosensor of this example, it is

8

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EP 0 502 504 A1

16

not necessary to distinguish a sample supply port
21 provided to the sensor from an air port 31.
Therefore, the sample supply port 21 can be used
as an air port, from which it is possible to supply
the sample liquid.

After forming the base 10 including the reac-
tion layer 15 and the hydrogen ion concentration
control layer 11 in the above-mentioned manner,
the base 10, a spacer 20 and a cover 30 were
laminated so as to be adhered as shown in Figure
6 with dashed lines.

The thus obtained fructose sensor was sup-
plied with 3 ul of a fructose standard solution as a
sample liquid from the sample supply port 21, and
the sensor response was measured in the same
manner as in Example 1 . The obtained result was a
proportional linear relation up to 25 mM of fructose.
Furthermore, the coefficient of variation (the CV
value) obtained in using 30 sensors was 6% or
less. Thus the repeatability was excellent.

Example 6

An electrode system 14 comprising a working
electrode 4 and counter electrode 5 was formed on
an insulating substrate 1 made from polyethylene
terephthalate by means of screen printing in the
same manner as in Example 1, and was ground
and heat treated. An aqueous solution including
potassium ferricyanide and CMC was dropped on
the electrode system 14 and dried by heating, to
form a layer including CMC and potassium fer-
ricyanide.

The time required for drying the layer depends
upon the heating temperature. Therefore, the cry-
stal diameter of the potassium ferricyanide con-
tained in the layer can be controlled by the heat
treatment condition. A shorter drying time makes
the crystal diameter of the potassium ferricyanide
smaller, therefore increasing solubility speed into
the sample liquid. In this way, the response speed
can be increased. On the other hand, when potas-
sium ferricyanide coexists with enzyme in the reac-
tion layer, the heating temperature can not be
freely set, because the enzyme activity is reduced
by heating at a high temperature.

Then, an ethanol solution including 0.5 wt% of
PVP was dropped so as to cover the entire layer
including potassium ferricyanide and CMC, and
was dried to form a PVP layer. Furthermore, a
fructose dehydrogenase solution was dropped on
the PVP layer, and was dried to form a fructose
dehydrogenase layer.

Then, an ethanol solution including 0.5 wt% of
hydroxyethyl cellulose so as to cover the entire
fructose dehydrogenase layer, and was dried to
form a hydroxyethyl cellulose layer. Furthermore, a
phosphate buffer solution (pH = 4.5) was dropped

on the hydroxyethyl cellulose layer, and was dried
to form a hydrogen ion concentration control layer
11. The thus obtained base 10, a spacer 20 and a
cover 30 were laminated in the same manner as in

5 Example 1 to form a fructose sensor.

PVP and hydroxyethyl cellulose are hydrophilic
polymer. Therefore, when an aqueous solution in-
cluding fructose dehydrogenase is dropped on the
PVP layer, the PVP layer and the fructose de-

70 hydrogenase layer are not completely mixed but
only mixed by a thickness of several microns on
the interface therebetween. Also when a phosphate
buffer solution is dropped on the hydroxyethyl cel-
lulose layer, the hydroxyethyl cellulose layer and

75 the hydrogen ion concentration control layer are
not completely mixed but only mixed by a thick-
ness of several microns on the interface there-
between.

In this way, the CMC-potassium ferricyanide

20 layer is separated from the fructose de-
hydrogenase layer and the hydrogen ion concen-
tration control layer respectively.

The fructose sensor produced in the above
method was supplied with 3 ul of a fructose stan-

25 dard solution from a sample supply port. The sen-
sor response after 2 minutes obtained in the same
manner as in Example 1 corresponded to the fruc-
tose concentration in the sample liquid. Alternately,
when the voltage was applied 90 seconds after the

30 supply, the response current was approximately
the same as that obtained 2 minutes after. This is
because potassium ferricyanide dissolved into the
sample liquid more quickly due to the smaller
crystal diameter of the potassium ferricyanide.

35 The response current values obtained by the

fructose sensor of this example were larger by
about 20% than those obtained by the comparative
fructose sensor without a hydrogen ion concentra-
tion control layer 11, as was in Example 1.

40 It is understood that various other modifications

will be apparent to and can be readily made by
those skilled in the art without departing from the
scope and spirit of this invention. Accordingly, it is
not intended that the scope of the claims appended

45 hereto be limited to the description as set forth
herein, but rather that the claims be construed as
encompassing all the features of patentable novelty
that reside in the present invention, including all
features that would be treated as equivalents there-

50 of by those skilled in the art to which this invention
pertains.

Claims

55 1. A biosensor for quantifying a substrate con-
tained in a sample liquid by reducing electron
acceptors using electrons generated in a reac-
tion of the substrate to enzyme and then by

9

17

EP 0 502 504 A1

18

measuring the reduced amount of the electron
acceptors electrochemicaliy, the biosensor
comprising:

an electrical insulating substrate, an elec-
trode system including at least a working elec-
trode and a counter electrode which are
formed on the insulating substrate, a reaction
layer including the enzyme provided on the
electrode system, and a hydrogen ion con-
centration control layer;

the reaction layer being in contact with the
electrode system.

2. A biosensor according to claim 1, wherein the
reaction layer further includes hydrophilic poly-
mer and electron acceptors.

3. A biosensor according to claim 1, wherein the
reaction layer further includes hydrophilic poly-
mer and the hydrogen ion concentration con-
trol layer includes electron acceptors.

4. A biosensor according to claim 2, wherein the
reaction layer is formed by laminating a first
layer including hydrophilic polymer and en-
zyme, a second layer including hydrophilic
polymer and a third layer including electron
acceptors in this order.

5. A biosensor according to claim 1, wherein the
hydrogen ion concentration control layer is
provided on the electrode system.

6. A biosensor according to claim 5, wherein the
reaction layer further includes hydrophilic poly-
mer and electron acceptors, and a hydrophilic
polymer layer is provided between the reaction
layer and the hydrogen ion concentration con-
trol layer.

7. A biosensor according to claim 5, wherein the
reaction layer comprises a first layer including
hydrophilic polymer and electron acceptors
and a second layer including enzyme lami-
nated on the first layer, and the hydrogen ion
concentration control layer is provided on the
second layer.

8. A biosensor according to claim 1, wherein the
electrode system is mainly formed from car-
bon.

9. A biosensor according to claim 1, wherein the
hydrogen ion concentration control layer in-
cludes buffering salt selected from the group
consisting of potassium biphosphate-dipotas-
sium phosphate, potassium biphosphate-dis-
odium phosphate, citric acid-disodium phos-

phate, citric acid-trisodium citrate, potassium
bicitrate-sodium hydroxide and maleic acid
monosodium salt-sodium hydroxide.

5 10. A biosensor according to claim 1, wherein the
hydrophilic polymer is selected from the group
consisting of carboximethyl cellulose, poly-
vinylpyrrolidone, hydroxyethyl cellulose,
hydroxy proryl cellulose, methyl cellulose,

io ethyl cellulose, ethyl hydroxyethyl cellulose,

carboxy methyl ethyl cellulose, polyvinyl al-
cohol and gelatin.

11. A biosensor according to claim 1, wherein the
75 enzyme is selected from the group consisting

of fructose dehydrogenase, invertase,
mutarotase, glucose oxidase, alcohol oxidase,
lactate oxidase, lactate dehydrogenase, choles-
terol oxidase, xanthine oxidase and amino acid
20 oxidase.

12. A biosensor according to claim 1, wherein the
electron acceptor is selected from the group
consisting of potassium ferricyanide, p-ben-

25 zoquinone, phenazinemethosulfate and fer-

rocene.

13. A method for producing a biosensor for quan-
tifying a substrate contained in a sample liquid

30 by reducing electron acceptors using electrons

generated in a reaction of the substrate to
enzyme and then by measuring the reduced
amount of the electron acceptors electrochemi-
caliy, the method comprising the steps of:

35 firstly, providing an electrode system in-

cluding at least a working electrode and a
counter electrode on an electrical insulating
substrate;

secondly, forming a hydrogen ion concen-
40 tration control layer including electron accep-

tors on the insulating substrate; and

finally, forming a reaction layer including
hydrophilic polymer and enzyme on the elec-
trode system.

45

50

55

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European Patent
Office

EUROPEAN SEARCH REPORT

Application Number

EP 92 10 3691

DOCUMENTS CONSIDERED TO BE RELEVANT

Category

Citation of document with indication, where appropriate,

Relevant

CLASSIFICATION OF THE

of relevant passages

to claim

APPLICATION ant. CI.S )

Y

EP-A-0 359 831 (MATSUSHITA ELECTRIC INDUSTRIAL

1.2.4.5,

C12Q1/00

CO.)

7-12

G01N27/404

* page 8, line 4 - page 10, line 20 *

page 11, Hne Zl - page 13, Hne 26 *

* page 15, line 23 - page 16, line 28 *

* page 27, Hne 2 - Hne 21 *

* page 32, Hne 2 - page 33, line 13 *

* claims 7,13,14; figures 4-6 *

A

3,6,13

Y

PATENT ABSTRACTS OF JAPAN

1.2.4,5.

vol. 13, no. 355 (P-914)9 August 1989

7-12

A

6,13

* abstract *

D

& JP-A-01 114 747 (MATSUSHITA ELECTRIC

INDUSTRIAL CO. ) 8 May 1989

A

EP-A-0 400 918 (NAKANO VINEGAR CO.)

1,11,12

* column 3, Hne 50 - column 5, Hne 27 *

* column 6, Hne 29 - column 9, Hne 55 *

* examples 8,10,14,17 *

TECHNICAL FIELDS

SEARCHED (Int. CI.S )

P. A

WO-A-9 109 139 (BOEH RINGER MANNHEIM)

1,2,5-7,

9-13

C12Q

page a, line s> — page iy, line Ji

G01N

* figures 1-3 *

The present search report has been drawn up for all claims

Place of tearca

BERLIN

Data of c»a«>l«tiaa at the Marc*

12 JUNE 1992

JOHNSON K.

S
fid
O

O
At
U

CATEGORY OF CITED DOCUMENTS

X : particularly relevant if taken alone

Y : particularly relevant if combined with another

document of the same category
A : technological background
O : non-written disclosure
P : intermediate document

T : theory or principle underlying the invention
E : earlier patent document, but published on, or

after the filing date
D : document cited in the application
L : document cited for other reasons

A : member of the same patent family, corresponding
document